1. Field of the Invention
The present invention relates to a method of acquiring an image and a scanning optical microscope for irradiating a sample emitting two or more types of fluorescences, namely, a multi-dye fluorescent sample with an excitation light as a light spot by condensing the light by an objective lens and detecting each fluorescence emitted from this sample by a photodetector through a pinhole, thereby acquiring an image of that sample.
2. Description of the Background Art
As a scanning optical microscope, there are known the following microscopes. One confocal pinhole is arranged at a position which is also conjugated by a sample. When the surface of the sample is scanned by a light spot, a plurality of rays of fluorescence are emitted from the sample. The rays of fluorescence emitted from the sample are led to the confocal pinhole. A plurality of the rays of fluorescence are subjected to optical path division by a dichroic mirror or a grating. A plurality of photodetectors corresponding to the respective rays of fluorescence are arranged to these optical paths, and a ray of fluorescence corresponding to each photodetector is detected (see Jpn. Pat. Appln. KOKAI Publication No. 8-43739).
When trying to simultaneously acquire a plurality of rays of fluorescence, a wavelength of fluorescence emitted from a fluorescent dye on a short wavelength side and a wavelength of fluorescence emitted from a fluorescent dye on a long wavelength side overlap each other. In a sample dyed using two or more kinds of fluorescent dyes, a fluorescent dye FITC is excited with an excitation wavelength of 488 nm, and emits a ray of fluorescence having a central wavelength of 520 nm. A fluorescent dye Cy5 is excited with an excitation wavelength of 633 nm, and emits a ray of fluorescence having a central wavelength of 670 nm. These wavelengths of fluorescence overlap each other and, as shown in FIG. 1, a phenomenon called xe2x80x9cfluorescent cross talkxe2x80x9d in which a ray of fluorescence on the short wavelength side (FITC) is mixed occurs in a detector configured to detect a ray of fluorescence on the long wavelength side (Cy5).
In order to avoid the fluorescent cross talk, there is known a technique for detecting the respective rays of fluorescence in the time division manner (see Jpn. Pat. Appln. KOKAI Publication No. 10-206745). This technique carries out switching of each excitation wavelength for exciting a sample dyed by two or more kinds of fluorescent dyes and a optical path to each detector configured to detect each ray of fluorescence in synchronization with light scanning.
At this time, switching of the excitation wavelength and the detection optical path is performed relative to a command for acquiring an image issued by a computer in accordance with one-frame scanning or each one line or during photo acceptance of one pixel, and each ray of fluorescence is detected in the time division manner, thereby acquiring an image. Further, a product catalogue of, for example, ZEISS Co. Ltd. discloses a product such that a galvanometer mirror on a high-speed scanning side which is light scanner reciprocates for scanning and the excitation wavelength is switched by an acousto-optic device (AOTF) for selecting a wavelength in accordance with an outward route and an inward route to detect different rays of fluorescence in the respective routes, thereby avoiding the fluorescent cross talk.
The confocal effect in the scanning optical microscope depends on dimensions of a diameter of a confocal pinhole and a diameter of a light spot (diffraction ray) according to a wavelength of each ray of fluorescence whose image is formed on the confocal pinhole.
That is, although it is ideal to reduce the diameter of the confocal pinhole in order to increase the resolution, an amount of fluorescence becomes extremely small. Therefore, the light which passes through the confocal pinhole and is detected becomes weak, and acquisition of an image with the excellent SN can not be expected. Thus, the dimension of the confocal pinhole diameter is matched with that of the diffraction diameter in order to optimize the brightness and the confocal effect in the direction of an optical axis. At this time, the dimension of a diffraction diameter d can be obtained by the following expression:
d=1.22xc2x7xcex/NA
xcex=central wavelength of a ray of fluorescence to be detected
NA=NA of a fluorescent light flux incident upon the confocal pinhole
In the above expression, the diameter of the confocal pinhole is matched with the diffraction diameter d obtained by substituting a fluorescent light wavelength xcex to be detected and NA of the fluorescent light flux incident upon the confocal pinhole determined by an objective lens.
In the above-described prior art technique, however, since a plurality of fluorescences emitted from the sample pass through one confocal pinhole, the diameter of the confocal pinhole can matched with only the fluorescence relative to one excitation wavelength. For example, when an excitation wavelength of 488 nm is used for excitation, the FITC emits a fluorescence having a central wavelength of 520 nm. Further, when an excitation wavelength of 633 nm is used for excitation, the Cy5 emits a fluorescence having a central wavelength of 670 nm. Therefore, assuming that NA of a fluorescence incident upon the confocal pinhole is 0.0063, the diameter of the confocal pinhole which is optimum for a fluorescence of FITC is as follows:
Moreover, the confocal pinhole diameter which is optimum for the fluorescent light of Cy5 is as follows:
Therefore, when the confocal pinhole diameter is set to 100 xcexcm, it is possible to obtain the confocal pinhole diameter which is optimum for the fluorescent light of FITC. However, for the fluorescent light of Cy5, the confocal pinhole diameter is too small, and a bright fluorescent image can not be obtained.
In addition, when the confocal pinhole diameter is set to 130 xcexcm, the confocal pinhole diameter which is optimum for the fluorescent light of Cy5 can be obtained. However, for the fluorescent light of FITC, the confocal pinhole diameter is too large, and the confocal effect is reduced.
In order to eliminate the above-described problems, there is disclosed a technique for optimizing the resolution and the brightness by matching the dimension of the pinhole diameter with the diffraction diameter by which the light from the sample is formed on the confocal pinhole plane (see Jpn. Utility Model Appln. KOKAI Publication No. 06-16927). By using this technique, an opening size (dimension of the pinhole diameter) of the confocal pinhole can be changed in accordance with an objective lens to be used or a wavelength to be observed.
As an adjustment mechanism for the opening size of the confocal pinhole, there is a method for performing. adjustment by arranging a plurality of pinholes on a concentric circle on a turret and rotating this turret (see Jpn. Utility Model Appln. KOKAI Publication No. 6-16927) or a method for performing adjustment by continuously moving and changing a pair of square openings each having a V shape by using a direct acting type motor (see Jpn. Pat. Appln. KOKAI Publication No. 2000-10152).
On the other hand, in a scanning optical microscope for observing fluorescences, a characteristic of a dichroic mirror for separating the illuminating lights to the sample and the fluorescences from the sample must be switched in accordance with the excitation wavelength of the sample to be observed or the fluorescent light spectral characteristic (see Jpn. Pat. Appln. KOKAI Publication No. 7-333508).
When this dichroic mirror is switched, since an image formation position on the confocal pinhole plane is shifted due to an error in a mounting angle or a difference in the parallelism of the dichroic mirror, the center of the confocal pinhole and the image formation position must be corrected by moving the optical axis or the confocal pinhole position.
As the correction method, there is, for example, a crisscross moving stage system using two motors for moving the confocal pinhole itself within the plane (see Jpn. Pat. Appln. KOKAI Publication No. 7-333508) or a system for matching the image formation position with the center of the confocal pinhole by rotating two parallel plane glasses by a motor and then moving the optical axis (see Jpn. Pat. Appln. KOKAI Publication No. 8-271792).
It is an object of the present invention to provide a method of acquiring an image and a scanning optical microscope by which the cross talk does not occur in all fluorescences and an optimum confocal effect can be obtained in accordance with each fluorescence to be detected when detecting a plurality of fluorescences.
It is another object of the present invention to provide a scanning optical microscope by which mechanical abrasion in a driving section does not occur and a speed of diameter correction by means for effectively restricting the diffraction diameter can be increased.
The present invention is characterized in that, when an excitation light having an excitation wavelength according to each fluorescent dye is switched and emitted with respect to a sample dyed with two or more kinds of fluorescent dyes in synchronization with light scanner and each fluorescence according to each excitation light is detected through one confocal pinhole in the time division manner to obtain one image, a pinhole diameter of the confocal pinhole is adjusted to a diameter suitable for the fluorescence emitted from the sample by the excitation light in accordance with the wavelength of the excitation light.
Specifically, the structure of the present invention is described as follows. It is to be noted that the xe2x80x9cpinholexe2x80x9d means a transmission type pinhole such that the fluorescence passes through the pinhole as well as a reflection type pinhole for reflecting the fluorescence such as a mirror having a pinhole shape.
According to the present invention, there is provided a scanning optical microscope comprising: a light source configured to selectively output an excitation light having an excitation wavelength according to each fluorescent dye to a sample dyed with two or more kinds of fluorescent dyes; scanner configured to scan the excitation light outputted from the light source; an objective lens configured to condense the excitation light scanned by the scanner onto the sample; a detector configured to detect a fluorescence having the fluorescent dye according to the excitation light by using the excitation light condensed by the objective lens; one confocal pinhole capable of adjusting a diameter of a pinhole arranged in front of the detector; controller configured to adjust a pinhole diameter of the confocal pinhole to a diameter suitable for the fluorescent rays emitted from the sample by the excitation light in synchronization with switching of the excitation light from the light source when each fluorescence according to each excitation light is detected in the time division manner through the confocal pinhole to acquire one image by changing over the excitation light with which the sample is irradiated in synchronization with scanning of the scanner. Preferred embodiments according to the present invention are as follows. It is to be noted that each of the following embodiments may be solely applied or combined and applied.
(1) The controller switches the excitation light in synchronization with scanning performed for each line relative to the sample by the light scanner.
(2) The controller switches the excitation light in synchronization with scanning performed for each frame relative to the sample by the light scanner.
(3) The controller switches the excitation light in units of pixel during scanning relative to the sample by the light scanner.
(4) The detector is configured by one detector.
(5) There is further included a barrier filter which is fixed in front of the detector, blocks two or more types of excitation lights for exciting the two or more types of fluorescences and transmits therethrough two or more types of fluorescences emitted from the sample.
(6) There are further included: a first barrier filter which blocks a first excitation light of the excitation lights and transmits therethrough fluorescences emitted from the sample by the first excitation light; a second barrier filter which blocks a second excitation light of the excitation lights and transmits therethrough fluorescences emitted from the sample by the second excitation light; and means for switching the first barrier filter and the second barrier filter between the confocal pinhole and the detector in synchronization with change of the excitation lights.
(7) The confocal pinhole is a minute device group having a plurality of minute devices, and a plurality of the minute devices are controlled by minute device controller.
(8) The confocal pinhole includes a minute device group configured by arranging a plurality of minute deflecting mirrors in the form of a two-dimensional matrix, and further includes minute device controller configured to control an angle of each minute deflecting mirror in the diffraction diameter in such a manner that the light spot is reflected in an arrangement direction of the photodetector and controlling an angle of each minute deflecting mirror outside the diffraction diameter to an angle different from that of each minute deflecting mirror in the diffraction diameter.
(9) In (7) or (8), the minute device group controller has a function for varying an area of each minute device for controlling to lead the light spot to the photodetector in accordance with the dimension of the diffraction diameter imaged to the minute device group.
(10) In (7) or (8), the minute device group controller has a function for correcting a central position of each minute device for controlling to lead the light spot to the photodetector in accordance with the displacement of the light spot imaged to the minute device group.
(11) In (10), there is provided a function for correcting the displacement of the light spot in the minute device group generated by switching of at least one optical device arranged between the sample and the minute device group.
(12) In (7) or (8), there are further included: a light source capable of selectively outputting to a sample dyed with two more types of fluorescent dyes an excitation light having an excitation wavelength according to each fluorescent dye; scanner configured to scan an excitation light outputted from the light source; and an objective lens configured to condense the excitation light scanned by the scanner, wherein the minute device group controller adjusts each minute device of the minute device group for leading a light from the sample to the photodetector to a diffraction diameter of a light spot imaged to the minute device group through the confocal lens in synchronization with switching of the excitation light from the light source when each fluorescence according to each excitation light is detected in the time division manner through one minute device group to acquire one image by changing over the excited lights with which the sample is irradiated in synchronization with scanning of the light scanner.
(13) In (12), switching of excitation lights by the minute device group controller is synchronized with scanning in accordance with each one line or scanning in an outward route and an inward route by the light scanner, respectively.
(14) In (12), switching of the excitation lights by the minute device group controller is synchronized with scanning in accordance with one frame by the light scanner.
(15) In (12), switching of the excitation lights by the minute device group controller is synchronized with scanning in accordance with one pixel by the light scanner.
Since the pinhole diameter is adjusted in accordance with a wavelength of a fluorescence emitted from the sample, there occurs no cross talk in all fluorescences when detecting a plurality of fluorescences, and an optimum confocal effect can be obtained in accordance with each fluorescence to be detected.
Further, since the minute deflecting mirror is adopted as the pinhole, mechanical abrasion of the driving section is not generated, and it is possible to speed up the diameter correction or the position correction of means for restricting the effective range of the diffraction diameter.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.